The overall goal of this project is to address the critical medical need for agents to combat antibacterial drug resistance by a novel approach of potentiating drug influx in Gram-negative non-fermenters such as Pseudomonas aeruginosa and Acinetobacter baumannii. These species exhibit intrinsic drug resistance due to the combined effects of a poorly permeable outer membrane and several multi-drug efflux pumps. The approach of this project is to develop novel adjunctive therapeutics to increase the intracellular effective levels of new and existing antibiotics. The OprF/OmpA family of porins in P. aeruginosa and A. baumannii appears to be the major route for influx of many existing antibiotics and likely for many new antibacterial since these porins are the major route for non-specific diffusion of drug-sized molecules across the outer membrane. However, these porins exist predominately (=95%) in a two-domain, closed-channel form, which spans the outer membrane and peptidoglycan layer to stabilize the cell structure. The low levels of one-domain, open- channel conformers reduce the outer membrane permeability by one to two orders of magnitude as compared to that of enterobacteriaciae such as Escherichia coli. The strategy of this proposal is to identify drug-like small molecules that shift the balance towards the open-channel porin conformer, thus improving antibacterial influx by opening the porin and destabilizing the bacterial cell structure. The two conformations are not in rapid equilibrium but appear to result from a structure-based bias for the two-domain, closed-channel from in the folding pathway of nascent proteins. Preliminary studies established that several specific mutations in P. aeruginosa OprF shift the ratio toward the open-channel form, and that a cysteine created at residue 312 in a form of OprF devoid of other cysteines is exposed on the cell surface only when OprF porins are in the single- domain, open-channel form. Labeling cys-312 with a fluorescent, membrane-impermeable dye provides an assay to detect and quantify open-channel forms. In Phase I, development and optimization of the assay will be completed to enable high throughput screening to identify small molecules that significantly shift the folding pathway toward open-channel conformers. The optimized screen will be applied to a diverse library of =300,000 discrete small molecules. Hits will be selected and confirmed in the screening assay and then validated for specificity and concentration-dependent potency in secondary assays including osmotic swelling rates of proteoliposomes containing OprF in the presence of L-arabinose, OprF protease sensitivity, and reduced MICs for cephalosporins in P. aeruginosa. Resulting non-cytotoxic, validated hits will be prioritized by their extent of potentiation of the MICs of a variety of antibacterials vs. multiple P. aeruginosa and A. baumannii clinical isolates as well as related species such as Burkholderia cepacia and Stenotrophomonas maltophilia. In Phase II, the most promising of these influx facilitators will be optimized to develop lead compounds for efficacy and toxicity testing in animal models.